Microfluidic tissue biopsy and immune response drug evaluation devices and systems

ABSTRACT

This disclosure describes microfluidic tissue biopsy and immune response drug evaluation devices and systems. A microfluidic device can include an inlet channel having a first end configured to receive a fluid sample optionally containing a tissue sample. The microfluidic device can also include a tissue trapping region at the second end of the inlet channel downstream from the first end. The tissue trapping region can include one or more tissue traps configured to catch a tissue sample flowing through the inlet channel such that the fluid sample contacts the tissue trap. The microfluidic device can also include one or more channels providing an outlet.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of application of U.S. Ser.No. 17/334,431, filed May 28, 2021 and titled “MICROFLUIDIC TISSUEBIOPSY AND IMMUNE RESPONSE DRUG EVALUATION DEVICES AND SYSTEMS,” whichis a divisional of and claims priority to application U.S. Ser. No.16/116,656, filed Aug. 29, 2018 and titled “MICROFLUIDIC TISSUE BIOPSYAND IMMUNE RESPONSE DRUG EVALUATION DEVICES AND SYSTEMS,” whichapplication claims priority to U.S. Provisional Patent Application62/552,264, filed Aug. 30, 2017 and titled “MICROFLUIDIC TISSUE BIOPSYAND IMMUNE RESPONSE DRUG EVALUATION DEVICES AND SYSTEMS,” and to U.S.Provisional Patent Application 62/581,667, filed Nov. 4, 2017 and titled“MICROFLUIDIC TISSUE BIOPSY AND IMMUNE RESPONSE DRUG EVALUATION DEVICESAND SYSTEMS,” all of which is incorporated herein by reference in theirentireties.

BACKGROUND

Current technology for simulating dynamic processes involvinginteractions between mammalian tissue samples and cells is gated by theinability to recapitulate the tissue microenvironment and interactionsbetween tissues, therapeutic compounds and the host immune system.

SUMMARY

One aspect of this disclosure is directed to microfluidic devicecomprising including a substrate. The substrate defines an inlet channelhaving a first end configured to receive a fluid sample optionallycontaining a tissue sample. The substrate defines a tissue trappingregion at the second end of the inlet channel downstream from the firstend. The tissue trapping region includes one or more tissue trapsconfigured to catch a tissue sample flowing through the inlet channelsuch that the fluid sample contacts the tissue trap. The substrate alsodefines one or more channels providing an outlet.

In some implementations, at least one of the one or more tissue trapscomprises an arrangement of one or more walls. In some implementations,the one or more channels providing the outlet include one or more branchchannels connecting to the second end of the inlet channel where thesecond end of the inlet channel and the tissue trapping region converge.In some implementations, the convergence of the second end of the inletchannel and the tissue trapping region further includes a first branchchannel coupled to the second end of the inlet channel at a firstjunction and configured to direct a first portion of the fluid sample ina first direction, and a second branch channel coupled to the second endof the inlet channel at the first junction and configured to direct asecond portion of the fluid sample in a second direction, different formthe first direction, wherein the tissue trap is positioned at the firstjunction.

In some implementations, the one or more channels providing the outletfurther include one or more suction channels downstream of the one ormore tissue traps and configured to hold the tissue sample in placewithin the one or more tissue traps. In some implementations, at leastone of the one or more tissue traps includes a bottom surface positionedat a lower depth than a bottom surface of the inlet channel. In someimplementations, the first branch channel and the second branch channelconverge at a second junction downstream from the one or more tissuetraps.

In some implementations, the microfluidic device further includes afirst suction channel coupling at least one of the one or more tissuetraps to the first branch channel at a third junction downstream fromthe second end of the inlet channel. The microfluidic device can alsoinclude a second suction channel coupling the at least one of the one ormore tissue traps to the second branch channel at a fourth junctiondownstream from the second end of the inlet channel. In someimplementations, a diameter of at least one of the one or more thetissue traps is about twice that of the inlet channel.

In some implementations, the tissue trapping region includes a ribbedchannel coupling the inlet channel to the one or more channels providingthe outlet. In some implementations, at least one of the one or moretissue traps can be defined by sidewalls of ribs of the ribbed channeland a bottom wall positioned at a lowest depth of the ribbed channel. Insome implementations, the at least one tissue trap can further includeat least a second tissue trap and a third tissue trap.

In some implementations, the tissue trapping region can include acircuitous channel having a first curved portion coupled to the secondend of the inlet channel. The microfluidic device can also include atleast one of the one or more tissue traps positioned at a center of thefirst curved portion such that the fluid sample flows along the firstcurved portion past the tissue trap. In some implementations, the one ormore channels providing the outlet channel can include a suction channelcoupling to the at least one of the one or more tissue traps andconfigured to carry the fluid sample downstream from the at least one ofthe one or more tissue traps. In some implementations, the circuitouschannel can further include a second curved portion coupled to adownstream end of the first curved portion and a second tissue trappositioned at a center of the second curved portion such that the fluidsample flows along the second curved portion past the second tissuetrap. In some implementations, a downstream end of the second curvedportion is coupled to the one or more channels providing the outlet.

In some implementations, the microfluidic device can also include aninlet port coupled to the first end of the inlet channel and configuredto deliver the fluid sample to the inlet channel. In someimplementations, the inlet port can include a first threaded connectorconfigured for attachment to a fluid line.

In some implementations, the microfluidic device can also include abubble trapping structure coupled to the inlet channel downstream fromthe inlet port. The bubble trapping structure can be configured tofacilitate evacuation of air bubbles from the fluid sample. In someimplementations, a surface of the bubble trapping structure can have ashape defined by a parabolic function. In some implementations, thebubble trapping structure can further include a second threadedconnector configured for attachment to an air release line.

In some implementations, the microfluidic device can also include anoutlet port coupled to the at least one of the one or more channelsproviding the outlet and configured to remove the fluid sample from themicrofluidic device. In some implementations, the substrate can beformed from a biocompatible material. In some implementations, thesubstrate can be formed from an optically transparent material, and themicrofluidic device can further include an optical interface providingoptical access to the tissue sample positioned within the tissuetrapping region. In some implementations, the one or more tissue trapscan be configured to entrain the tissue sample in place within the oneor more tissue traps.

Another aspect of this disclosure is directed to a method for evaluatingan interaction between a tissue sample and a fluid sample. The methodcan include introducing a tissue sample into a first end of an inletchannel of a microfluidic device. The method can include introducing afluid sample into the first end of the inlet channel to cause the tissuesample to flow to a tissue trapping region at a second end of the inletchannel downstream from the first end. The tissue trapping region caninclude a tissue trap configured to catch the tissue sample such that atleast a portion of the fluid sample contacts the tissue sample. Themethod can include collecting the sample fluid from at least one channelproviding an outlet downstream from the tissue trapping region.

In some implementations, the method can include priming the inletchannel with fluid prior to introducing the tissue sample into the firstend of the inlet channel. In some implementations, the method caninclude observing an interaction between the tissue sample and the fluidsample in the tissue trapping region. In some implementations, themicrofluidic device can be formed from a transparent material, andobserving the interaction between the tissue sample and the fluid samplecan further include positioning a lens of a microscope in proximity tothe microfluidic device.

In some implementations, the tissue trap can be configured to secure thetissue sample without damaging the tissue sample. In someimplementations, the method can include introducing the tissue samplevia a bubble trapping structure coupled to the inlet channel, andintroducing the fluid sample via an inlet port coupled to the inletchannel. The inlet port can be upstream from the bubble trappingstructure. In some implementations, the method can include removing airfrom the fluid sample via the bubble trapping structure.

In some implementations, the method can include releasing the tissuesample from the tissue trap by introducing a second fluid sample into atleast one of the one or more channels configured to provide the outletsuch that the second fluid sample flows towards the inlet channel.

In some implementations, after collecting the sample fluid at least oneof the one or more channels configured to provide the outlet downstreamfrom the tissue trapping region, the method can include reintroducingthe collected sample fluid into the inlet channel of the microfluidicdevice.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. Likereference numbers and designations in the various drawings indicate likeelements. For purposes of clarity, not every component may be labeled inevery drawing.

FIG. 1A illustrates a perspective view of an example microfluidicdevice, according to an illustrative implementation.

FIG. 1B illustrates a second perspective view of the examplemicrofluidic device of FIG. 1A, according to an illustrativeimplementation.

FIG. 2A illustrates a cross-sectional view of a portion of an examplemicrofluidic device that can be used to implement the microfluidicdevice of FIG. 1A, according to an illustrative implementation.

FIG. 2B illustrates a tissue sample within the microfluidic device ofFIG. 2A, according to an illustrative implementation.

FIG. 2C is a visual depiction of the flow characteristics of themicrofluidic device of FIG. 2A, according to an illustrativeimplementation.

FIG. 2D illustrates a first arrangement of the microfluidic device ofFIG. 2A having suction channels, according to an illustrativeimplementation.

FIG. 2E illustrates a second arrangement of the microfluidic device ofFIG. 2A having suction channels, according to an illustrativeimplementation.

FIG. 2F illustrates a third arrangement of the microfluidic device ofFIG. 2A having suction channels, according to an illustrativeimplementation.

FIG. 3A illustrates a cross-sectional view of a portion of an examplemicrofluidic device that can be used to implement the microfluidicdevice of FIG. 1A, according to an illustrative implementation.

FIG. 3B illustrates a perspective view of the portion of themicrofluidic device shown in FIG. 3A, according to an illustrativeimplementation.

FIG. 3C is a visual depiction of the flow characteristics of themicrofluidic device of FIG. 3A, according to an illustrativeimplementation.

FIG. 4A illustrates a cross-sectional view of a portion of an examplemicrofluidic device that can be used to implement the microfluidicdevice of FIG. 1A, according to an illustrative implementation.

FIG. 4B is a visual depiction of the flow characteristics of themicrofluidic device of FIG. 4A, according to an illustrativeimplementation.

FIG. 4C illustrates a first arrangement of the microfluidic device ofFIG. 4A having suction channels, according to an illustrativeimplementation.

FIG. 4D is a visual depiction of the flow characteristics of themicrofluidic device of FIG. 4C, according to an illustrativeimplementation.

FIG. 5 illustrates a bubble trapping structure that can be included inthe microfluidic device of FIG. 1A, according to an illustrativeimplementation.

FIG. 6 illustrates a flowchart of a method for evaluating an interactionbetween a tissue sample and a fluid sample, according to an illustrativeimplementation.

DETAILED DESCRIPTION

This disclosure aims to establish a robust platform to recapitulate thetissue microenvironment and interactions with host immune cells.

This disclosure describes devices and systems capable of recapitulatingthe tissue microenvironment and tissue interactions with fluid which maycontain cells (such as circulating immune cells), medications,therapeutic compounds, or other components. As used herein, “fluid” canrefer to fluid containing components that are intended to interact witha tissue sample (such as cells, medications, therapeutic compounds, orother substances) in order to observe a response, or can refer to fluiddevoid of such components. A key challenge in this regard is the abilityto maintain a tissue sample, such as a tumor biopsy, in a configurationthat permits real-time observation of tumor viability and responses totherapeutic compounds, such as dynamic interactions between circulatingimmune cells and the tissue biopsy sample. This disclosure describesmultiple novel designs capable of capturing and maintaining the positionof a tissue sample in a flow field that presents cells, medications,therapeutic compounds, or other components to the tissue in aphysiologically relevant manner, permitting control over perfusion ratesand shear forces to ensure that results are relevant to human in vivoconditions.

Beyond the tissue trapping and flow field device, in order to fullyrecapitulate the dynamics of tissue interactions with cells such asimmune cells, medications, therapeutic compounds, or other components,and to do so in a high throughput manner, it can be useful to integratethe device with a system capable of sustaining the tissue, maintainingcontrol over the flow rate, viability of cells and density ofcirculating components, and to avoid problems common to microfluidicsystems such as bubbles, debris, blockages or variability in flow rates.A key challenge is the ability to integrate these features in a mannerthat provides robust control over system dynamics for periods of up toone week or more.

In some implementations, the devices of this disclosure are capable ofex vivo simulation of the dynamics of tissue interactions with variousfluid components, such as cells, medications, or therapeutic compounds.The devices can integrate capture regions, cell flow channels,resistance lines and fluidic connections, and bubble trappingstructures. The devices described herein can permit observation andcontrol over interactions between various types of fluid components andexcised tissues such as tumor biopsy samples, skin biopsies, epithelialtissues such as gut, airway, renal or reproductive tract tissues. Thefigures and corresponding description below provide further detailedinformation regarding the design of such devices and systems. In brief,this disclosure includes various aspects, including specific designs fortissue traps, including a heart-shaped branching structure, ribbedchannel bottom structure, S-curve structure, and suction port structure.Each serves as a means to precisely control and freeze the position of atissue biopsy sample in a flow stream, and to expose the fixed tissuesample to a precisely controlled flow of fluid containing componentssuch as cells, medications, or therapeutic compounds in order to observeinteractions between the fluid components and tissue samples. Thisdisclosure also includes aspects relating to integration of thesetrapping devices with other fluidic components. These additionalcomponents can include resistance channels, fluidic connectors andbranch points, tissue sample loading ports, bubble trapping structures,drug dosing and media sampling ports, cell containment vessels, andmanifolds that serve as distribution branches for cells and gas pressurelines.

For tissue trapping regions, other ways to address the problem includethe use of V-shaped posts to trap tissues, side chamber regions, orside-to-side channels with cells flowing through one lane and tissuesheld in another, with a gel region in between. Additional potentialdesigns for these systems include methods where the biopsied tissuesample is contained within a side channel or side compartment thatindirectly receives flow from the main dynamic perfusion channel,methods where excised biopsy samples are contained within larger excisedtissues or organs, or methods where biopsy samples are contained withinconstructs that are molded from mammalian tissues.

In other implementations, interactions between fluid and tissues can bemimicked by generating tissue constructs contained within gel or matrixregions. Fluid can flow through adjacent channels in which they arepermitted to migrate toward the matrix-embedded tissue constructs. Somesuch devices and systems can utilize conventional microwell plates ortranswells to contain excised tissues, as a static representation of thecell-tissue interaction.

The devices of this disclosure include innovative aspects in the natureof the tissue trapping geometry as compared to other approaches that mayuse V-shaped posts, side chambers, or side-to-side channels withintervening gel regions. The disadvantages of these approaches relate tothe inability to precisely control the rate at which circulating fluidare presented to the tissue biopsy sample, because V-shaped post regionsrequire dealing with a tradeoff between allowing flow around the tissueand raising the hydrostatic pressure of flow against the tissue sample.For side chambers or side-to-side channels, tissue interactions withfluid can occur via migration phenomena, which may be difficult tocontrol in the microenvironment, or by random “strikes” of fluidtraveling obliquely through the flow stream. This disclosure providesnovel designs that can be used to contain tissue biopsy samples andchannels for flowing fluid.

Other approaches to solving the tissue-cell interaction problem includeusing conventional means to contain tissues and fluid (e.g., staticwells or transwells) and/or gel-matrix systems in which tissue samplesare disaggregated and seeded into microfluidic devices in compartmentsadjacent to blood/cell-flow channels. Technical obstacles to theinnovations described herein include developing designs capable ofcapturing tissue biopsy samples and effectively causing interaction ofthese captured samples with flowing media. These innovative concepts arenot obvious because they include new tissue biopsy sample containmentdesigns that overcome previous limitations. Key advantages of thedevices and systems of this disclosure include designs that effectivelyentrain tissue biopsy samples and expose them to flowing fluid in amanner that optimizes the cross-section of interaction between the two.

FIG. 1A illustrates a perspective view of an example microfluidic device100, according to an illustrative implementation. FIG. 1B illustrates asecond perspective view of the example microfluidic device 100 of FIG.1A. Similar reference numerals in FIGS. 1A and 1B refer to similarelements. Referring to both FIG. 1A and FIG. 1B, the microfluidic device100 can be used to simulate interactions between tumors or other tissuesamples and the immune system, for example by providing amicroenvironment for testing the effectiveness of immunotherapytreatments on lymphocytes and tumor biopsies taken directly from apatient. As a result, the microfluidic device 100 can be used to modelthe in vivo environment and analyze the prolonged response of a tumorand circulating lymphocytes to the controlled introduction ofimmunotherapy pharmaceuticals. Thus, the microfluidic device 100 canenables judicious administration of immunotherapy treatments by allowingmedical professionals to make informed decisions regarding course oftreatment for a patient based on experiments conducted using themicrofluidic device 100.

The microfluidic device 100 is formed from a substrate 102. Thesubstrate 102 defines a variety of structural features, including aninlet port 105 leading to an inlet channel 115. Downstream from theinlet port 105 and coupled to the inlet channel 115 is a bubble trappingstructure 110. Farther downstream from the inlet channel 115 is a tissuetrapping region 120, which leads to an outlet channel 125. An outletport 130 is positioned at a downstream end of the outlet channel 125.While only a single microfluidic device 100 is depicted in FIGS. 1A and1B, it should be understood that in some implementations, multipledevices similar to the microfluidic device 100 can be incorporated intoa single chip without departing from the scope of this disclosure.

In use, the microfluidic device 100 can capture a tissue sample andallow testing of the interaction of the tissue sample with variouscells, medications, therapeutic compounds, or other agents or componentsincluded within a fluid sample flowing within the microfluidic device100. For example, a tissue sample, such as a portion of a tumor, can beloaded into the device via the inlet port 105 or via the bubble trappingstructure 110. After the tissue sample flows through the inlet channel115, the structural characteristics of the tissue trapping region 120cause the tissue sample to become trapped. A fluid sample can then beintroduced into the inlet port 105 and flowed through the inlet channel115, while the tissue sample remains held in place in the tissuetrapping region 120. At least a portion of the fluid sample (and thecells, medications, therapeutic compounds, or other components withinthe sample) can contact the trapped tissue sample as it flows from theinlet channel 115 to the outlet channel 125 and finally exits themicrofluidic device 100 via the outlet port 130. In someimplementations, air bubbles that may be present in the fluid sample,and which may cause damage to the tissue sample or may otherwiseinterfere with the results of the experiment, can be removed from themicrofluidic device 100 via the bubble trapping structure 110.

It should be understood that, in the implementation shown in FIG. 1A,the outlet channel 125 serves as an outlet for the microfluidic device100 as a whole, but not for the tissue trapping region 120. Thus, insome implementations, the outlet channel 125 may not be an outletchannel relative to the tissue trapping region 120, and therefore may bereferred to by a different name. In some implementations, one or morechannels may provide an outlet for fluid at or near the tissue trappingregion 120. For example, branching channels, suction channels, and otherchannels further described below may provide such an outlet. Thus, insome implementations, these channels also may be referred to as outletchannels. Various types of channels that may provide an outlet for fluidat or near the tissue trapping region 120 are described further below.

In some implementations, the microfluidic device 100 can be furtherconfigured to provide an optical interface for viewing the interactionsite where the tissue sample interacts with the fluid sample. Tofacilitate optical access, the channels within the microfluidic device100 can be configured to substantially avoid optical distortion. In someimplementations, the channels can have a rounded rectangularcross-sectional shape. Such a shape exhibits smaller surface area tovolume ratio than a purely rectangular channel, which can help topreserve pumping efficiency by reducing resistance in the channels. Inaddition, rounded rectangular channels may not produce image distortionthat is characteristic of channels having circular cross-sectionalshapes.

These and other aspects of this disclosure are described further below.In particular, a variety of different geometries and structural shapescan be used to implement the tissue trapping region 120, and severalexamples of such geometries are shown in the figures. In particular,FIGS. 2A-2F generally relate to a first geometry for the tissue trappingregion 120, FIGS. 3A and 3B generally relate to a second geometry forthe tissue trapping region 120, and FIGS. 4A-4D generally relate to athird geometry for the tissue trapping region 120.

FIG. 2A illustrates a cross-sectional view of a portion of an examplemicrofluidic device 200 that can be used to implement the microfluidicdevice 100 of FIG. 1A, according to an illustrative implementation. Thefeatures of the microfluidic device 200 generally correspond to thefeatures of the microfluidic device 100, and like reference numeralsrefer to like elements. For example, the microfluidic device 200includes an inlet channel 215, a tissue trapping region 220, and anoutlet channel 225 that can carry fluid out of the microfluidic device200. FIG. 2 shows the structural details of the tissue trapping region220, which in this example includes a tissue trap (also referred to as atissue trapping zone or trapping zone) 235 positioned at a downstreamend of the inlet channel 215, as well as two branch channels 240 a and240 b branching off from the inlet channel 215 in opposing directions ata junction near the tissue trap 235.

As described above, the tissue trapping region 220 is configured to trapa tissue sample in a fixed location while a fluid sample is flowedthrough the microfluidic device 200. For example, in someimplementations, the tissue trapping region 220 is shaped such that,when the fluid sample flows through the microfluidic device 200, astagnation zone exists in at least a portion of the area of the tissuetrap 235, causing the tissue sample to become trapped in the tissue trap235. FIG. 2B illustrates a tissue sample 239 within the microfluidicdevice 200 of FIG. 2A, according to an illustrative implementation. Itshould be noted that FIG. 2B shows the microfluidic device 200 in areversed orientation relative to that shown in FIG. 2A, such that fluidflows from right to left in the depiction of the microfluidic device 200of FIG. 2B. As shown, the tissue sample 239 becomes trapped in thetissue trap 235 in a manner that allows the fluid sample to continueflowing through the inlet channel 215 to the branch channels 240 a and240 b, while a portion of the fluid sample contacts the tissue sample239 as it flows.

In some implementations, the tissue trap or trapping zone 235 can have abottom wall that is positioned at a lower depth than the bottom of theinlet channel 215 that leads up to it. That is, the tissue trap 235 canbe stepped down relative to the bottom surface of the inlet channel 215.Thus, the tissue trap 235 can serve as a pocket for catching, trapping,holding, immobilizing, or securing the tissue sample 239. In someimplementations, the shape of the tissue trapping region 220, includingthe tissue trap 235, is selected to catch or otherwise facilitatetrapping of the tissue sample 239 while the fluid sample passes throughthe microfluidic device 200. For example, the tissue trap 235 can have adiameter that is larger than that of the inlet channel 215. In someimplementations, the tissue trap 235 can have a diameter that is abouttwice that of the inlet channel 215. FIG. 2C is a visual depiction 252of the flow characteristics of the microfluidic device 200 of FIG. 2A,according to an illustrative implementation. The shading within thechannels shows the velocity of the streamlines within the device. Whenthe streamlines bend at the branch channels 240 a and 240 b, the inertiaof the tissue sample can overcome the viscous forces and can becomelodged in the tissue trap 235.

Referring again to FIG. 2B, the trapping of the tissue sample 239 in amanner that allows the fluid sample to continue flowing through thedevice while contacting the tissue sample 239 can allow the interactionsbetween the tissue sample 239 and agents within the fluid sample. Forexample, in some implementations fluorescent materials can be added toeither the fluid sample or the tissue sample 239, and the visualcharacteristics of the tissue sample 239 and the fluid sample can beobserved over time. To facilitate such observation, the microfluidicdevice 200 can be formed from a material that is transparent andoptically clear, at least in the region of the device near the tissuetrap 235. This area can serve as an optical interface that can beexamined by an optical instrument, such as a camera or a microscope,that is brought into proximity with the microfluidic device 200.

FIG. 2D illustrates a first arrangement 201 of the microfluidic deviceof FIG. 2A having suction channels, according to an illustrativeimplementation. Components shown in the arrangement 201 aresubstantially similar to the components shown in FIG. 2A, and likereference numerals refer to like elements. However, the arrangement 201of FIG. 2D differs from that shown in FIG. 2A in that the arrangement201 includes a suction channel 245. The suction channel 245 is coupledbetween a downstream end of the tissue trap 235 and the outlet channel225. Thus, the suction channel 245 can provide an outlet for fluid inthe tissue trap 235, and therefore may sometimes itself be referred toas an outlet channel. Similarly, the microfluidic device 201 alsoincludes branch channels 240 a and 240 b that can provide an outlet forfluid near the tissue trap 235, and therefore the branch channels 240 aand 240 b may also be referred to as outlet channels 240 a and 240 b.Furthermore, it should be understood that the outlet channel provides anoutlet of the microfluidic device 201 (i.e., it is configured to carryfluid out of the microfluidic device 201), but does not couple to thetissue trap 235 and therefore does not serve as an outlet for fluid fromthe tissue trap 235. In some implementations, the suction channel 245can be configured to facilitate trapping of the tissue sample within thetissue trap 235. For example, as the fluid sample flows from left toright in the depiction of FIG. 2D, through the branch channels 240 a and240 b and into the outlet channel 225, the suction channel 245 cancreate a pressure drop or suction effect that tends to cause the tissuesample to be forced towards the right-hand side of the tissue trap 235,thereby becoming lodged within the tissue trap 235 more forcefully.

FIG. 2E illustrates a second arrangement 202 of the microfluidic deviceof FIG. 2A having suction channels, according to an illustrativeimplementation. Components shown in the arrangement 202 aresubstantially similar to the components shown in FIG. 2A, and likereference numerals refer to like elements. However, the arrangement 202of FIG. 2E differs from that shown in FIG. 2A in that the arrangement202 includes two suction channels 245 a and 245 b. The suction channels245 a and 245 b are coupled between a downstream end of the tissue trap235 and the branch channels 240 a and 240 b, respectively. In someimplementations, the suction channels 245 a and 245 b can be configuredto facilitate trapping of the tissue sample within the tissue trap 235,in a manner similar to that of the suction channel 245 shown in FIG. 2D.For example, as the fluid sample flows from left to right in thedepiction of FIG. 2E, through the branch channels 240 a and 240 b, thesuction channels 245 a and 245 b can create a pressure drop or suctioneffect that tends to cause the tissue sample to be forced towards theright-hand side of the tissue trap 235, thereby becoming lodged withinthe tissue trap 235 more forcefully. In addition, because the suctionchannels 245 a and 245 b couple directly to a downstream end of thetissue trap 235, the suction channels 245 a and 245 b can provide anoutlet for fluid in the tissue trap 235. Therefore, in someimplementations the suction channels 245 a and 245 b may sometimes bereferred to as outlet channels.

Similarly, FIG. 2F illustrates a third arrangement 203 of themicrofluidic device of FIG. 2A having suction channels 245 a and 245 b,according to an illustrative implementation. The arrangement 203 of FIG.2F is similar to the arrangement 202 of FIG. 2E, with the exception thatthe suction channels 245 a and 245 b in the arrangement 203 couple to ajunction of the branch channels 240 a, 240 b, and the outlet channel225. However, the suction channels 245 a and 245 b in the arrangement203 serve a similar purpose to that described above in connection withFIG. 2E. That is, as the fluid sample flows from left to right in thedepiction of FIG. 2F, through the branch channels 240 a and 240 b andinto the outlet channel 225, the suction channels 245 a and 245 b cancreate a pressure drop or suction effect that tends to cause the tissuesample to be forced towards the right-hand side of the tissue trap 235,thereby becoming lodged within the tissue trap 235 more forcefully. Thesuction channels 245 a and 245 b couple directly to a downstream end ofthe tissue trap 235, thereby providing an outlet for fluid in the tissuetrap 235. Therefore, in some implementations the suction channels 245 aand 245 b may sometimes be referred to as outlet channels.

FIG. 3A illustrates a cross-sectional view of a portion of an examplemicrofluidic device 300 that can be used to implement the microfluidicdevice of FIG. 1A, according to an illustrative implementation. FIG. 3Billustrates a perspective view of the portion of the microfluidic device300 shown in FIG. 3A. The features of the microfluidic device 300generally correspond to the features of the microfluidic device 100, andlike reference numerals refer to like elements. For example, themicrofluidic device 300 includes an inlet channel 315, a tissue trappingregion 320, and an outlet channel 325. FIGS. 3A and 3B show thestructural details of the tissue trapping region 320, which in thisexample includes a ribbed channel coupled between the inlet channel 315and the outlet channel 325. The ribbed channel includes ribs, such asthe ribs 355 a-355 c (generally referred to as ribs 355), that projectinto the ribbed channel. The ribbed channel also defines tissue traps335 a-335 c (generally referred to as tissue traps 335).

In general, each of the tissue traps 335 has sidewalls defined by asubset of the ribs 355. As shown, the bottom wall of each tissue trap335 is positioned at a lowest depth of the ribbed channel, which islower than the bottom wall of the inlet channel 315 and the outletchannel 325. While the depiction of FIG. 3A shows the ribbed channeldefining three tissue traps 355, it should be understood that, in otherimplementations, the ribbed channel may include any number of ribs 355defining any number of tissue traps 335 without departing from the scopeof this disclosure.

Similar to the tissue trapping region 220 shown in FIG. 2A, the tissuetrapping region 320 (including the tissue traps 335) can be configuredto trap a tissue sample in a fixed location while a fluid sample isflowed through the microfluidic device 300. For example, the tissuetrapping region 320 is shaped such that, when the fluid sample flowsthrough the microfluidic device 300, the tissue sample becomes trappedin the tissue traps 335. In some implementations, a separate tissuesample can become trapped in each of the tissue traps 335. In some otherimplementations, one or more of the tissue traps 335 may remain unusedfor a given experiment.

In some implementations, the ribbed shape of the tissue trapping region320, including the tissue traps 335, is selected to facilitate trappingof a tissue sample while the fluid sample passes through themicrofluidic device 300. FIG. 3C is a visual depiction 352 of the flowcharacteristics of the microfluidic device 300 of FIG. 3A, according toan illustrative implementation. The shading within the channels showsthe velocity of the streamlines within the microfluidic device 300.Generally, a tissue sample will be larger and heavier than otherparticles that flow through the device 300 within the fluid sample. As aresult, the tissue sample will tend to sink within the flow due togravity. Thus, positioning the tissue traps 335 at the lowest depth ofthe ribbed channel, which includes small obstructing ribs 355, can helpto cause the tissue sample to become trapped within one of the tissuetraps 335.

It should be understood that the microfluidic device 300 can include anyof the features and functionality described above with respect to themicrofluidic device 100 and the microfluidic device 200 shown in FIGS.1A and 2A, respectively. For example, the microfluidic device 300 can beformed from a material that is transparent and optically clear in theregion of the device near the tissue traps 335, which can serve as anoptical interface that can be examined by an optical instrument broughtinto proximity with the microfluidic device 300. As a result, the tissuesamples and the fluid sample in the tissue traps 335 can be observedoptically over time.

It should be understood that, in the implementation shown in FIG. 3A,the outlet channel 325 serves as an outlet for the microfluidic device100 as a whole, and also for the tissue trapping region 120. In someimplementations, although not illustrated in FIG. 3A, the microfluidicdevice 300 also may include one or more additional channels that serveas outlets for fluid at or near the tissue trapping region 320, whichmay also be referred to as outlet channels. For example, such channelsmay be branch channels or suction channels similar to those describedabove in connection with FIGS. 2D-2F.

FIG. 4A illustrates a cross-sectional view of a portion of an examplemicrofluidic device 400 that can be used to implement the microfluidicdevice 100 of FIG. 1A, according to an illustrative implementation. Thefeatures of the microfluidic device 400 generally correspond to thefeatures of the microfluidic device 100, and like reference numeralsrefer to like elements. For example, the microfluidic device 400includes an inlet channel 415, a tissue trapping region 420, and anoutlet channel 425. FIG. 4A shows the structural details of the tissuetrapping region 420, which in this example includes a circuitous channelcoupled between the inlet channel 415 and the outlet channel 425. Thecircuitous channel includes a first curved portion 460 a and a secondcurved portion 460 b (generally referred to as curved portions 460). Thecurvature of the first curved portion 460 a is opposed to the curvatureof the second curved portion 460 b. The first curved portion 460 aincludes a first tissue trap 435 a positioned at its center. The secondcurved portion 460 b is coupled to a downstream end of the first curvedportion 460 a, and includes a second tissue trap 435 b positioned at itscenter. The first tissue trap 435 a and the second tissue trap 435 b aregenerally referred to as tissue traps 435 in this disclosure. Thedownstream end of the second curved portion 460 b is coupled to theoutlet channel 425.

While the depiction of FIG. 4A shows the circuitous channel as includingtwo curved portions 460 a and 460 b, it should be understood that, inother implementations, the circuitous channel may include any number ofcurved portions each defining a respective tissue trap 435 withoutdeparting from the scope of this disclosure. For example, the circuitouschannel may include only a single curved portion (i.e., the first curvedportion 460 a), or may include three or more curved portions.

Similar to the tissue trapping regions 220 shown in FIG. 2A and 320shown in FIG. 3A, the tissue trapping region 420 (including the tissuetraps 435) can be configured to trap a tissue sample in a fixed locationwhile a fluid sample is flowed through the microfluidic device 400. Forexample, the tissue trapping region 420 is shaped such that, when thefluid sample flows through the microfluidic device 400, a respectivetissue sample can become trapped in the tissue traps 435. In someimplementations, a separate tissue sample can become trapped in each ofthe tissue traps 435. In some other implementations, one or more of thetissue traps 435 may remain empty.

In some implementations, the circuitous shape of the tissue trappingregion 420, including the tissue traps 435, is selected to facilitatetrapping of a tissue sample while the fluid sample passes through themicrofluidic device 300. FIG. 4B is a visual depiction 452 of the flowcharacteristics of the microfluidic device 400 of FIG. 4A, according toan illustrative implementation. The shading within the channels showsthe velocity of the streamlines within the microfluidic device 400.Generally, a particle (such as a tissue sample) in the fluid sample willtend to follow the streamline located at its center of mass. If theReynolds number of the tissue sample is sufficiently large, the inertiaof the particle will overcome the viscous forces when the streamlinesbend along the circuitous path including the curved portions 460 of thetissue trapping region 420. As a result, the tissue sample will tend tobecome secured within the one of the tissue traps 435.

FIG. 4C illustrates a first arrangement 401 of the microfluidic device400 of FIG. 4A having a suction channel, according to an illustrativeimplementation. Components shown in the arrangement 401 aresubstantially similar to the components shown in FIG. 4A, and likereference numerals refer to like elements. However, the arrangement 401of FIG. 4C differs from that shown in FIG. 4A in that the arrangement401 includes only a single tissue trap 435 a, as well as a suctionchannel 465. The suction channel 465 is coupled between the tissue trap435 a and the outlet channel 425. In some implementations, the suctionchannel 465 can be configured to facilitate trapping of the tissuesample within the tissue trap 435 a. For example, as the fluid sampleflows from left to right in the depiction of FIG. 4D, into the outletchannel 425, the suction channel 465 can create a pressure drop orsuction effect that tends to cause the tissue sample to be forcedtowards the right-hand side of the tissue trap 435 a, thereby becominglodged within the tissue trap 435 a more forcefully. FIG. 4D illustratesthe flow characteristics of the microfluidic device 401 of FIG. 4C,according to an illustrative implementation. As described in the flowcharacteristic figures above, the shading in FIG. 4C shows the velocityof the streamlines within the microfluidic device 401. In addition,because the suction channel 465 couples directly to a downstream end ofthe tissue trap 435 a, the suction channel 465 can provide an outlet forfluid in the tissue trap 435 a. Therefore, in some implementations thesuction channel 465 may sometimes also be referred to as an outletchannel.

FIG. 5 illustrates a bubble trapping structure 110 that can be includedin the microfluidic device 100 of FIG. 1A, according to an illustrativeimplementation. Generally, the bubble trapping structure 110 can help tofacilitate the capture of air bubbles from within the fluid sample thatflows through the microfluidic device 100, whose presence may beundesirable. Bubbles can be introduced into the microfluidic device 100,for example, during the tissue loading process or via the incoming flowof the fluid sample. In some implementations, bubbles can negativelyimpact experimental outcomes. Therefore, it may be desirable to preventair bubbles from entering the system, or to remove them before theyreach the tissue sample downstream. Incorporation of an in-line bubbletrapping structure 110 into the microfluidic device 100 allows for easyremoval of air introduced by either mechanism

As shown, the microfluidic device 100 is coupled to a ceiling of theinlet channel 115. The bubble trapping structure 110 includes sidewallsthat curve inwards toward each other in a direction away from the inletchannel 115. As shown in FIG. 1A, the bubble trapping structure 110 canbe positioned downstream from the inlet port 105, such that air bubblesintroduced through the inlet port 105 can be removed via the bubbletrapping structure 110 before they reach the tissue trapping region 120.In some implementations, the shape of the sidewalls of the bubbletrapping structure 110 can be defined by a parabolic function. Themicrofluidic device 100 also includes a threaded connector 510. Thethreaded connector 510 can be configured for attachment to an air line,through which air bubbles can be removed from the device after beingcaptured by the bubble trapping structure 110.

The bubble trapping structure 110 is incorporated directly into themicrofluidic device 100. This design eliminates the need for an externalair removal device, thereby reducing the number of required connections.Additionally, inclusion of the bubble trapping structure 110 within themicrofluidic device 100 can reduce the overall fluid volume requirement.In some implementations, the bubble trapping structure 110 can beconfigured to produce limited disruption of the primary flow path of thefluid sample through the inlet channel 115. For example, the paraboliccurvature of the bubble trapping structure 110 can encourage the gentleremoval of bubbles from the flow, and the threaded connector 510, whichcan couple to an air line or a syringe, allows evacuation of air fromthe chimney as needed.

In some implementations, the bubble trapping structure 110 also can beconfigured to serve as the loading port for the tissue sample. Forexample, the opening of the bubble trapping structure 110 can beconfigured to accommodate a pipette tip through which the tissue sampleis introduced into the microfluidic device 100. In some implementations,the tissue sample can be injected through the bubble trapping structure110, which may include a valve that can be closed after that tissuesample is injected. Flow of the fluid sample from the inlet port 105 canthen cause the tissue sample to flow towards the tissue trapping region120, where it becomes secured in place as described above.

FIG. 6 illustrates a flowchart of a method 600 for evaluating aninteraction between a tissue sample and a fluid sample, according to anillustrative implementation. In some implementations, the method 600 canbe carried out using a microfluidic device such as the microfluidicdevice 100 shown in FIG. 1A. In brief overview, the method 600 caninclude introducing a tissue sample into an inlet channel of amicrofluidic device (step 605), introducing a fluid sample into theinlet channel to cause the tissue sample to flow to a tissue trappingregion of the microfluidic device (step 610), collecting the samplefluid from one or more channels providing an outlet downstream from thetissue trapping region (step 615), and observing an interaction betweenthe tissue sample and the fluid sample in the tissue trapping region(step 620).

Referring again to FIG. 6 , the method 600 can include introducing atissue sample into an inlet channel of a microfluidic device (step 605).In some implementations, the tissue sample can be or can include aportion of a tumor or other cancerous cells whose reaction to animmunotherapy is of interest. The tissue sample can be injected into themicrofluidic device, for example via a port configured to serve as abubble trapping structure similar to that shown in FIG. 5 . In someimplementations, the inlet channel can first be primed with a fluidbefore the tissue sample is introduced. This can allow the tissue sampleto be introduced directly into a fluid, which may help to betterpreserve the tissue sample for experimentation.

The method 600 also can include introducing a fluid sample into theinlet channel to cause the tissue sample to flow to a tissue trappingregion of the microfluidic device (step 610). In some implementations,the fluid sample can include cells, medications, therapeutic compounds,or other components. In some implementations, the fluid sample can beintroduced at an area of the inlet channel upstream from the area wherethe tissue sample was introduced. For example, referring to themicrofluidic device 100 of FIG. 1A, the tissue sample can be introducedvia the bubble trapping structure 110, and the fluid sample can beintroduced at the inlet port 105, upstream from the bubble trappingstructure 110. This tissue sample and fluid sample introductiontechnique can help to ensure that the fluid sample is able to carry thetissue towards the tissue trapping region, which can be downstream fromthe areas in which both the fluid sample and the tissue sample areintroduced.

In some implementations, the tissue trapping region can include at leastone tissue trap configured to trap the tissue sample. The tissue trapcan include an intersection or junction of one or more fluidly connectedchannels, cavities, spaces, or chambers. In some implementations, thegeometry of the tissue trap can result in a stagnation zone configuredsuch that the fluid flow characteristics in the stagnation zone arerelatively stagnant (i.e., fluid velocity is lower, and in some casesmay be zero) as compared with the fluid flow characteristics of otherportions of the microfluidic device.

In some implementations, the tissue trap can be positioned at anintersection of a relatively large inlet channel and one or morerelatively smaller branching channels that carry fluid away from thetissue trap to an outlet channel, for example as illustrated by thetissue trap 235 shown in FIG. 2A. Other structural features also maycontribute to the functionality of the tissue trap. For example, in someimplementations the tissue trap can include an elevation change relativeto the channels that couple to it, such that tissue trap serves as asunken pocket for receiving and securing the tissue sample. As a result,in some implementations, the tissue trap may sometimes be referred to asa tissue trapping pocket. In some implementations, other walls of thetissue trap also may be stepped away, stepped up or stepped down fromthe walls of channels that lead to them. For example, a ceiling of thetissue trap may be positioned at an elevated height relative to theceiling of the inlet channel, and the sidewalls of the tissue trap maybe farther apart from one another than the sidewalls of the inletchannel.

In addition, the branching channels carrying fluid away from the tissuetrap, as well as the outlet channel, can have a size that helps to trapthe tissue sample within the tissue trap. For example, the branchingchannels and the outlet channel can be sized such that tissue sampleslarger than about 300 microns cannot progress to the outlet of themicrofluidic device from the tissue trap. Thus, the tissue sample canbecome secured within the tissue trap, such that the cells in the fluidsample can contact the tissue sample as the fluid sample flows throughthe microfluidic device.

In some implementations, the tissue trap or trapping zone can have ageometry that is selected and/or arranged to trap the tissue samplewithout damaging the tissue sample. The tissue trap or trapping zone maybe formed in any geometrical shape or combination of geometries. Thetissue trap may be formed as a chamber or portion of a chamber and insome implementations may be referred to as a trapping chamber. Thetissue trap may be formed as any type of pocket, such as a partialpocket or a covered pocket, and in some implementations may be referredto as a trapping pocket. The tissue trap may be formed as any type ofcavity and may be referred to as a trapping cavity in someimplementations. The tissue trap may be designed, configured and formedsuch as to provide a pressure drop or suction effect with respect tofluid sample flows traversing an opening of the tissue trap and in someimplementations, may be referred to as a pressure drop trap, suctiontrap or tissue pressure drop zone or tissue suction zone.

The tissue trap may be formed as an arrangement of one or more walls.The one or more walls may be selected designed or configured withpredetermined heights and/or lengths and/or widths, such as in relationto any of the dimensions of the device comprising the tissue trap. Theone or more walls may be formed to meet at predetermined angles and/orpredetermined points, such as in relation to any of the dimensions orgeometries of the device comprising the tissue trap. The one or wallsmay be formed to be at predetermined orientations with respect to otherwalls and/or other walls of the device comprising the tissue trap. Forexample, the tissue trap can include one or more walls configured tosecure the tissue sample. The walls may be formed from the edges ofchannels that are in fluid communication with the tissue trap, or may beformed from the edges of the tissue trap itself In some implementations,a wall included in a tissue trap can be a sidewall, a bottom surface, ora ceiling. In some implementations, the tissue trap may include a curvedwall, or may include two or more substantially flat walls that couple toone another at an edge. A wall included in a tissue trap can beconfigured to restrict the motion of a tissue sample without shearing,tearing, or otherwise damaging the tissue sample, in contrast to othertypes of structures that may be designed to trap a tissue sample. Forexample, while a series of narrow posts may be used to secure a tissuesample at a particular point within a microfluidic device, therelatively small width of such posts relative to the width of the tissuesample can cause the tissue sample to become torn by the posts as fluidpressure is exerted on the tissue sample by the fluid flowing throughthe device. Because a wall has a larger surface area than such a post,the tissue traps described in this disclosure can secure a tissue samplewhile substantially reducing the risk that the tissue sample will becometorn or damaged.

In some implementations, a tissue trap also may include one or morechannels, such as suction channels, that exit from a rear surface of thetissue trap and join with branching channels and or an outlet channeldownstream from the tissue trap. Examples of such suction channels areillustrated in by the suction channels 240 a and 240 b of FIGS. 2D-2Fand the suction channel 465 of FIG. 4C. As fluid flows through themicrofluidic device, such suction channels can cause a pressure drop orother suction force to more securely trap a tissue sample within thetissue trap. Thus, in some implementations, the tissue trap may bereferred to as a suction trap. Examples of suitable geometries for sucha tissue trap have been described above, for example in connection withFIGS. 1A, 2A, 3A, and 4A.

The method 600 also can include collecting the sample fluid from one ormore channels providing an outlet downstream from the tissue trappingregion (step 615). In some implementations, the microfluidic device caninclude an outlet port coupled to an outlet channel and configured toallow the fluid sample to be collected. For example, the outlet port caninclude a threaded connector, which can be coupled to a fluid line or asyringe to extract the fluid sample. In some implementations, the airbubbles also can be extracted from the fluid sample. For example, airbubbles can be extracted via a bubble trapping structure such as thebubble trapping structure 110 shown in FIG. 5 . In some implementations,the bubble trapping structure can be positioned upstream from the tissuetrapping region, such that air bubbles can be extracted from the fluidsample before they reach the tissue trapping region.

In some implementations, the method 600 also can include reintroducingthe collected sample fluid into the inlet channel of the microfluidicdevice. That is, the fluid sample can be recirculated one or more timesthrough the microfluidic device. For example, the fluid sample can beintroduced into the microfluidic device at step 610 and can be collectedat step 615. Then, the same fluid sample can be recirculated through themicrofluidic device by reintroducing the fluid sample back into theinlet channel of the microfluidic device, and again collecting the fluidsample from the one or more channels providing the outlet. In someimplementations, steps 610 and 615 of the method 600 can be iterated anynumber of times.

The method 600 also can also include observing an interaction betweenthe tissue sample and the fluid sample in the tissue trapping region(step 620). Because the microfluidic device as described in thisdisclosure can be configured to simulate the dynamics of tissue-cellinteractions that occur in vivo, the observation of the interactionbetween the tissue sample and the fluid sample can provide valuableinsights into the way in which a patient will respond to a particularimmunotherapy. In some implementations, the microfluidic device can beformed form a transparent and/or optically clear material, and can besufficiently thin to permit observation of the interaction between thetissue sample and the fluid sample by external equipment. For example,the microfluidic device can include an optical interface positioned nearthe tissue trapping region, to allow a microscope, camera, or otheroptical equipment to be used to observe the interaction that takes placein the tissue trapping region from outside of the microfluidic device.In some implementations, at least one the tissue sample and the fluidsample can include fluorescent particles that may be observed by suchoptical equipment.

In some implementations, the method 600 also can include releasing thetissue sample from the tissue trap. To release the tissue sample, insome implementations a second fluid sample can be introduced into theone or more channels providing the outlet. This can cause the secondfluid sample to flow towards the inlet channel. This reverse flow offluid can exert fluid forces on the tissue sample within the tissue trapthat tend to dislodge the tissue sample from the tissue trap. In someimplementations, the tissue sample may be brought to an inlet port ofthe microfluidic device in this manner, and may be collected and removedfrom the device at the inlet port.

Having now described some illustrative implementations, it is apparentthat the foregoing is illustrative and not limiting, having beenpresented by way of example. In particular, although many of theexamples presented herein involve specific combinations of method actsor system elements, those acts and those elements may be combined inother ways to accomplish the same objectives. Acts, elements andfeatures discussed only in connection with one implementation are notintended to be excluded from a similar role in other implementations.

The systems and methods described herein may be embodied in otherspecific forms without departing from the characteristics thereof. Theforegoing implementations are illustrative rather than limiting of thedescribed systems and methods. Scope of the systems and methodsdescribed herein is thus indicated by the appended claims, rather thanthe foregoing description, and changes that come within the meaning andrange of equivalency of the claims are embraced therein.

1. A microfluidic device comprising: a substrate defining: an inletchannel having a first end configured to receive a fluid sampleoptionally containing a tissue sample; a tissue trapping region at thesecond end of the inlet channel downstream from the first end, thetissue trapping region including one or more tissue traps configured tocatch a tissue sample flowing through the inlet channel such that thefluid sample contacts the tissue trap; and one or more channelsproviding an outlet.